This grant proposal addresses the role of Rip2 in Huntington's disease (HD) pathology. Rip2 modulates a number of activities of relevance to disease pathogenesis in HD. Rip2 activates caspase-1, as well as regulates a number of other critical intracellular signaling pathways. By analyzing brains from R6/2 transgenic mice that model HD, we showed that levels of Rip2 increase progressively as the animals' condition worsens. We observed parallel trends in post mortem brain samples from patients with increasingly advanced HD. In Specific Aim 1, we will repeat our experiments using YAC128 mice, animals expressing the entire mutant human HD gene and not just a fragment thereof. Besides determining the time course of Rip2 expression, we will investigate consequent changes in the molecular and cellular biology of HD mice. Measurements of the enzyme activity of caspases and the posttranslational modifications of Bcl-2-family proteins will be compared between HD mice and HD mice in which the Rip2 gene has been ablated. Finally, we will measure disease endpoints, i.e., body weight, motor control, time of disease onset, and mortality, during the course of neurodegeneration. Specific Aims 2 and 3 more directly investigate the molecular mechanism by which Rip2 protein modulates neuronal cell death. Aim 2 concerns changes in Rip2 protein's intracellular localization consequent upon its posttranslational modification by small ubiquitin-like modifier (SUMO). We will measure the relative extent of sumoylation of Rip2 from nuclear and cytoplasmic fractions of HD and healthy brain tissue. Using mass spectrometry, we will identify the specific amino acid residues within Rip2 which become sumoylated. We will mutate these residues to determine if sumoylation is required for nuclear localization of Rip2 and mutant Htt-mediated cell death. Aim 3 concerns the role of Rip2 in the epigenetic gene regulation in HD. We will study the regulation of epigenetic pathways as a putative mechanism by which Rip2 affects HD. Finally, we will determine if posttranslational modification of Rip2 is required for epigenetic gene regulation and subsequent neuronal death in HD using HD mice lacking expression of Rip2.